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PubMed2006

Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia.

During encystation Giardia trophozoites secrete a fibrillar extracellular matrix of glycans and cyst wall proteins on the cell surface. The cyst wall material is accumulated in encystation-specific vesicles (ESVs), specialized Golgi-like compartments generated de novo, after export from the endoplasmic reticulum (ER) and before secretion. These large post-ER vesicles neither have the morphological characteristics of Golgi cisternae nor sorting functions, but may represent an evolutionary early form of the Golgi-like maturation compartment. Because little is known about the genesis and maturation of ESVs, we used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on two-dimensional electrophoresis gels, i.e. several proteasome subunits and HSP70-BiP. We show that BiP is exported to ESVs and retrieved via its C-terminal KDEL signal from ESVs. In contrast, cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to ESVs during encystation. This suggests that maturation of bulk exported cyst wall material in the Golgi-like ESVs involves both continuous activity of ER-associated quality control mechanisms and retrograde Golgi to ER transport.

Animals↗

PubMed1998

The last few milliseconds in the life of a secretory granule. Docking, dynamics and fusion visualized by total internal reflection fluorescence microscopy (TIRFM).

We have monitored single vesicles (granules) in bovine adrenal chromaffin cells using an optical sectioning technique, total internal reflection fluorescence microscopy (TIRFM). With TIR, fluorescence excitation is limited to an optical slice near a glass/water interface. In cells located at the interface, granules loaded with fluorescent dye can be visualized near to or docked at the plasma membrane. Here we give evidence that (1) TIRFM resolves single vesicles and (2) the fluorescence signal originates from vesicles of roughly 350 nm diameter, presumably large dense core vesicles (LDCVs). (3) Diffusional spread of released vesicle contents can be resolved and serves as a convenient criterion for a fusion event. (4) We give details on vesicle properties in resting cells, such as lateral mobility of chromaffin granules, number density, and frequency of spontaneous fusion or withdrawal into the cytoplasm. (5) Upon stimulation with high extracellular potassium, TIRFM reports depletion of the 'visible pool' of vesicles closest to the plasma membrane within hundreds of milliseconds, consistent with previous concepts of a release-ready pool. We conclude that TIRFM constitutes an independent assay for pool depletion. TIRFM will allow us to study aspects of secretion that have previously been inaccessible in living cells, in particular the spatial relations and dynamics of vesicles prior to and during exocytosis and re-supply of the near-membrane pool of vesicles.

Animals↗

PubMed1986

Subcellular localization of transferrin protein and iron in the perfused rat liver. Effect of Triton WR 1339, digitonin and temperature.

The subcellular localization of 3H-labelled 59Fe-loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques. More than 96% of the 59Fe present in the liver of rats perfused with 59Fe-labelled transferrin is recovered in the parenchymal cells. Rat livers were perfused with 10 micrograms/ml 3H-labelled 59Fe-saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant (S) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients. 3H label distributes essentially around densities of 1.13-1.14 g/ml overlapping to a large extent with the distribution of galactosyltransferase, the marker enzyme of the Golgi complex. However, after treatment with low concentrations of digitonin the 3H label dissociates from galactosyltransferase and is shifted to higher densities, suggesting an association of transferrin with cholesterol-rich endocytic vesicles which could derive from the plasma membrane. 59Fe is mostly found in the supernatant fraction largely in the form of ferritin, as indicated by its reaction with antiferritin antibodies. In the mitochondrial fraction the density distribution of 59Fe suggests an association with lysosomes and/or mitochondria. In contrast to the lysosomal enzyme cathepsin B, the density distribution of 59Fe was only slightly affected by pretreatment of the rats with Triton WR 1339, suggesting its association with the mitochondria. At 15 degrees C, 59Fe and 3H labels are recovered together in low-density endocytic vesicles. On the basis of our results we suggest that, at low extracellular transferrin concentration, iron uptake by the liver involves endocytosis of the transferrin protein. The complex is interiorized in low-density acidic vesicles where iron is released. The iron passes into the cytosol, where it is incorporated into ferritin and into the mitochondria. The iron-depleted transferrin molecule would then be returned to the extracellular medium during the recycling of the plasma membrane.

Animals↗

PubMed1985

X-ray microanalysis of mineralized matrix vesicles of experimental saccular aneurysms.

An energy dispersive X-ray microanalytical study was designed to examine the mineral deposits in matrix vesicles found in the walls of experimental aneurysms from two rabbits (103 and 1071 days postoperatively) and two sheep aneurysms (234 and 1202 days postoperatively). The freeze-substitution technique was adopted for use to retain inorganic ions in situ. Numerous, various sized extracellular electron-dense structures, believed to be matrix vesicles were observed. Size histograms for the mineralized vesicles showed that the proportion of smaller vesicles was higher in the older animals. A total of 370 vesicles were analyzed. Calcium and phosphorus with small amounts of magnesium were identified. No particular calcium phosphate mineral was dominant with the mean Ca/P molar ratio for all animals falling in the 1.1-1.2 range. Correlation coefficients for interrelationships between calcium, phosphorus, magnesium, and size were weak except for calcium vs phosphorus which was close to one, consistent with some type of calcium phosphate being the major constituent of the mineralized vesicles. Smaller electron-dense particles, probably mitochondrial granules were seen in some smooth muscle cells; a small number were analyzed and contained calcium and phosphorus (mean Ca/P molar ratio of 0.86) but no magnesium.

Aneurysm↗

PubMed2002

Vesicular transport of extracellular acid phosphatases in yeast Saccharomyces cerevisiae.

A method for isolation of secretory vesicles from the yeast Saccharomyces cerevisiae based on the disintegration of protoplasts by osmotic shock followed by separation of the vesicles by centrifugation in a density gradient of Urografin was developed in this study. Two populations of the secretory vesicles that differ in density and shape were separated. Acid phosphatases (EC 3.1.3.2) were used as markers of the secretory vesicles. It was shown that the constitutive acid phosphatase (PHO3 gene product) is mainly transported to the cell surface by a lower density population of vesicles, while the repressible acid phosphatase (a heteromer encoded by PHO5, PHO10, and PHO11 genes) by a vesicle population of higher density. These data provide evidence that at least two pathways of transport of yeast secretory proteins from the place of their synthesis and maturation to the cell surface may exist. To reveal the probable reasons for transport of Pho3p and Pho5p/Pho10p/Pho11p enzymes by two different kinds of vesicles, we isolated vesicles from strains that synthesize the homomeric forms of the repressible acid phosphatase. It was demonstrated that glycoproteins encoded by the PHO10 and/or PHO11 genes could be responsible for the choice of one of the alternative transport pathways of the repressible acid phosphatase. A high correlation coefficient between bud formation and secretion of Pho5p phosphatase and the absence of correlation between bud formation and secretion of minor phosphatases Pho10p and Pho11p suggests different functional roles of the polypeptides that constitute the native repressible acid phosphatase.

Acid Phosphatase↗

PubMed2001

Novel dual 'small' vesicle model of ATP- and noradrenaline-mediated sympathetic neuromuscular transmission.

The main question asked was if sympathetic nerves in guinea-pig vas deferens release the co-transmitters ATP and noradrenaline from the same, or different vesicles, i.e. in fixed combinations or independently. The extracellularly recorded excitatory junction current (EJC) and the fractional increase in overflow of tritium (delta T) were used to monitor the per pulse secretion of ATP and [3H]NA, respectively, during electrical stimulation with 1-3000 pulses at 0.1-40 Hz. The frequency- and train length-dependence and alpha 2-adrenoceptor-mediated autoinhibition of these parameters, and of the ATP-mediated twitch contraction, were compared first in the presence of cocaine (to block noradrenaline reuptake), then after brief exposure to phenoxybenzamine (PBA, to irreversibly 'destroy' alpha 2-autoreceptors). Parallel variations of EJC/p(ulse) and delta T/p(ulse) under all conditions would support, non-parallel variations argue against exocytosis of ATP and noradrenaline from the same vesicles. The main findings were that facilitation and alpha 2-autoinhibition of EJC/p and delta T/p were remarkably similar during stimulation at 2 Hz but increasingly dissimilar at higher frequencies. delta T/p remained strongly facilitated and tightly controlled by activation of alpha 2-autoreceptors at 10-40 Hz, but both the facilitation and the sensitivity to alpha 2-autoinhibition of EJC/p were inversely related to frequency. At 40 Hz EJCs were 'small', minimally facilitated and totally unaffected by cocaine or PBA, i.e. insensitive to alpha 2-autoinhibition. Nevertheless, activation of alpha 2-receptors during the 40 Hz train strongly restricted the 'post-tetanic augmentation' (PTA) of the first EJC 10 s after the tetanus. Comparison between the frequency dependence of EJCs and the twitch contraction in the presence of cocaine or after PBA treatment indicates that it is the 'summed EJC per second', i.e. the ATP-driven current injection per unit time into smooth muscle, which triggers the twitch. The working hypothesis is proposed that these nerves use two classes of 'small vesicles' (SVs) to store and release either 'big' or 'small' ATP and noradrenaline 'quanta', and that differences in properties (Ca2+ affinity, capacity) of Ca2+ receptors in the SV membranes enable the nerves to selectively secrete 'big quanta' at low frequency and 'small quanta' during trains at high frequency.

Action Potentials↗

PubMed1971

Fine structure of the medial nucleus of the trapezoid body of the bat with special reference to two types of synaptic endings.

The medial nucleus of the trapezoid body has been studied electron microscopically in two species of bat, Miniopterus schreibersi fuliginosus and Vespertilio superans, which were perfused with three different kinds of fixatives, osmium tetroxide, glutaraldehyde, and formaldehyde. Two types of synaptic endings are observed in the nucleus: the abundant calyciferous endings and the less frequently occurring "small-vesicle endings." The former endings vary greatly in size, and contain extended extracellular spaces between pre- and post-synaptic membranes. The latter endings are always small, without the extended extracellular spaces, and tend to lie side by side. In all of the materials perfused with three different fixatives, synaptic vesicles in the calyciferous endings are round in shape and larger than those in the small-vesicle endings. The shape of vesicles in the small-vesicle endings varies according to the kinds of fixatives used; round in osmium tetroxide-fixed materials, flattened in formaldehyde-fixed materials, and somewhat round or flattened in glutaraldehyde-fixed materials. It is suggested that the calyciferous endings are excitatory in nature and that the small-vesicle endings are inhibitory.

Aldehydes↗

PubMed1988

Morphological and immunohistochemical characteristics of a cell line originated from noninvasive human bladder transitional-cell carcinoma.

A cell line (IG) was derived from a noninvasive transitional cell carcinoma grade II. The cells were propagated in vitro for 9 months, had a low proliferative capacity, formed tumor nodules, and showed four characteristic morphological stages. Primary epithelial-like cells were characterized as stage I; fibroblast-like cells with loss of contact inhibition of growth as stage II; fibroblast-like cells with long cytoplasmic projections, massive glycosaminoglycan content and tumor nodule formation as stage III; and, as stage IV, granulated stellate cells. The cell culture progressed cytologically from well-differentiated stage I to less-differentiated stage IV. The four stages were confirmed and examined by electron microscopy. Immunoreactivity for fibronectin, keratin, epithelial membrane antigen and carcinoembryonic antigen shown by the cell line and the tissue indicates that the cell line represents the original cell population in the tumor. Glycosaminoglycan production by these cells was found to be responsible for the formation of tumor nodules which appeared in culture at the same time as the patient had developed an invasive bladder tumor. Glycosaminoglycans were observed as a massive accumulation intracellularly and extracellularly, as rods or thick slices in layers of adherent vesicles and granules covering the cell culture. Normal bladder epithelial cells and fibroblasts grew in monolayer, showed contact inhibition of growth, were devoid of intracellularly accumulated glycosaminoglycans and produced uniform extracellular matrix different from that of IG cells.

Adult↗

PubMed1980

Fibronectin presence in native collagen fibrils of human fibroblasts: immunoperoxidase and immunoferritin localization.

Fibronectin is a major constituent of the fibroblast extracellular matrix. Fibronectin binds to collagen, mediates fibroblast adhesion to collagen, and is synthesized and secreted into the medium of cultured fibroblasts. Affinity-purified antibodies to fibronectin and collagen were localized using the peroxidase-antiperoxidase method or with ferritin-coupled secondary antibodies. Using human fibroblasts cultured under routine conditions, fibronectin and procollagen I react in a nonperiodic manner with: 1) approximately 10 nm extracellular fibrils, 2) cell membrane, and 3) membrane-associated vesicles. All fibrils react with both antibodies, suggesting some form of codistribution of fibronectin and collagen in these fibrils. Treatment with ascorbate leads to the development of a larger diameter extracellular fibril, approximately 40 nm in diameter. These large diameter fibrils are clearly collagen fibrils as documented by the procollagen antibody reaction. Importantly, fibronectin is bound to or a constituent of these "native" or cellular made collagen fibrils. Fibronectin and procollagen antibodies localized with the peroxidase-antiperoxidase method have a 70 nm axial repeat of reaction product on ascorbate-treated fibroblasts. Localization of antibodies with ferritin-labeled secondary antibodies is less satisfactory, but supports the basic observations made with the unlabeled antibody enzyme method. This observation rules out any potential criticisms. Although it is more difficult to observe with immunoferritin, there is an indication that antibodies to fibronectin react with an axial periodicity on cellular produced collagen fibrils.

Collagen↗

PubMed1985

Ca2+ metabolism during cholinergic stimulation of acid secretion.

Carbachol-induced changes in 45Ca fluxes and cytosolic free Ca2+ were characterized in rabbit gastric glands and isolated dog parietal cells. Cholinergic stimulation was expressed as changes in the membrane permeability of the parietal cell to Ca2+ without Ca2+ release from intracellular stores. The changes in the membrane permeability to Ca2+ were sustained as long as the cells were exposed to carbachol, independent of extracellular Ca2+, and had the properties of a passive pathway across which Ca2+ distributed according to its chemical gradient. As a consequence of the changes in membrane permeability to Ca2+, carbachol caused a sustained increase in free cytosolic Ca2+ from a resting level of 134 +/- 11 to 533 +/- 81 nM (n = 14). After carbachol stimulation, calcium ions were sequestered in mitochondrial and probably nonmitochondrial compartments. When cell stimulation was terminated by atropine or La3+, the cells restored the resting level of the intracellular Ca2+ activity ([Ca2+]in), independent of uncoupling of mitochondrial oxidation phosphorylation. Reduction in [Ca2+]in was mainly through Ca2+ efflux across the plasma membrane of the parietal cell. Manipulation of intracellular Na+ ion activity in intact cells and studies with basal-lateral membrane vesicles from gastric mucosa indicated the absence of extracellular Na+-intracellular Ca2+ exchange activity in the plasma membrane of the parietal cell. A calmodulin-regulated, ATP-dependent Ca2+ pump that could maintain active Ca2+ extrusion from parietal cells was found in the basal-lateral membrane of the parietal cells.

Animals↗

PubMed1980

Influence of tetracycline on the calcification of epiphyseal rat cartilage. Transmission and scanning electron-microscopic studies.

Tetracyclines are known to interfere with bone calcification. We therefore studied their effects on matrix vesicle production and initial calcification of cartilage. 15-day-old rats were injected intraperitoneally with oxytetracycline 100 mg/kg, 6 consecutive injections every 12 h. Epiphyseal plates were examined by scanning and transmission electron microscopy and compared to light microscopy findings. It was found that high doses of tetracyclines cause degeneration of the chondrocytes in the proliferating and hypertrophic zones. Chondrocytes had short processes with only few matrix vesicles covering their surface. In the chondrocytic lacunae of the longitudinal septa of hypertrophic and calcifying cartilage there were fewer matrix vesicles as compared to controls, and their ability to aggregate and form mineralized calcospherites was impaired. This was further proven when bones were immersed in 7% NaOCl or ashed, as minerals containing calcospherites were hardly seen. It is therefore presumed that interference with intracellular and probably extracellular accumulation of calcium by tetracycline might inhibit matrix vesicle production and aggregation, thus inhibiting calcification.

Animals↗

PubMed1995

A method to trap IgG antibodies within functional membrane vesicles.

Monoclonal antibodies have been used extensively to study the roles of specific extracellular proteins in cellular activation. Due to the large size of these molecules, their use for the study of receptor-mediated activation of intracellular processes has been limited. This report describes a method to introduce whole IgG antibody molecules into large functional membrane vesicles (ghosts) derived from rat basophilic leukemia (RBL) cells. Furthermore, an IgG1 mouse monoclonal antibody (JRK) directed against the cytoplasmic portion of the beta subunit of the high affinity receptor for IgE (Fc epsilon RI) can partially inhibit Fc epsilon RI-mediated PI hydrolysis through modest effects on both basal and receptor-mediated activation of phospholipase C.

Animals↗

PubMed1994

Localization and putative function of the UL20 membrane protein in cells infected with herpes simplex virus 1.

The UL20 protein of herpes simplex virus 1, an intrinsic membrane protein, is required in infected Vero cells in which the Golgi apparatus is fragmented for the transport of virions from the space between the inner and outer nuclear membranes and for the transport of fully processed cell membrane-associated glycoproteins from the trans-Golgi to the plasma membrane. It is not required in the human 143TK- cell line, in which the Golgi apparatus remains intact. We report the following. (i) The UL20 protein was detected in infected cells beginning at 6 h postinfection and was regulated as a gamma 1 gene. (ii) Pulse-chase experiments revealed no detectable alteration in the mobility of the UL20 protein in polyacrylamide gels. (iii) In both infected Vero and infected 143TK- cells, the UL20 protein was detected by immunofluorescence in association with nuclear membranes and in the cytoplasm. Some of the cytoplasmic fluorescence colocalized with beta-COP, a protein associated with Golgi-derived transport vesicles. UL20 protein was present in virions purified from the extracellular space but could not be detected in the plasma membrane. These results are consistent with the hypothesis that UL20 is a component of virion envelopes and membranes of virion transport vesicles and is selectively retained from the latter in a Golgi compartment.

Animals↗

PubMed1996

Influence of specimen preparation on the identification of phospholipids by the phospholipase A2-gold method in mineralizing cartilage and bone.

The role of phospholipids in biological mineralization has been hypothesized but not fully elucidated. In order to identify phospholipids at the ultrastructural level in the mineralizing extracellular matrix, rat epiphyseal cartilage and metaphyseal bone have been labeled with the phospholipase A(2) (PLA(2))-gold method. The specificity and the efficiency of phospholipid detection have been evaluated by postembedding labeling of sections from epoxy- or hydrophilic resin-embedded samples, and by preembedding labeling of cryosectioned samples. The efficiency of the labeling was higher in cryosections than in hydrophilic resin-embedded specimens, while lower efficiency was found in epoxy resin-embedded samples. A 2- to 6-fold increase of the labeling density in calcified with respect to uncalcified areas of cartilage and bone has been found, depending on the specimen preparation used. The labeling intensity was significantly higher, at the periphery of the calcifying nodules in the epiphyseal cartilage matrix and in the calcifying osteoid, while the fully calcified bone matrix presented a weak labeling. Matrix vesicles, which are considered a possible source of extracellular phospholipids, appeared labeled in cryosections and in epoxy resin-embedded samples after a preincubation with PLA(2), which also increased the labeling of the intracellular membranes. The localization of phospholipids in the areas of initial mineralization suggests some hypotheses on the possible involvement of these molecules in the mineral phase deposition process.

Animals↗

PubMed1983

Morphological studies of neurotransmitter release and membrane recycling in sympathetic nerve terminals in culture.

The morphological correlates of transmitter release from synapses and varicosities were examined in mature cultures of sympathetic neurons dissociated from neonatal rat superior cervical ganglia. The number of synaptic vesicles decreased in synapses and varicosities depolarized with 53 mM K+. The decrease in vesicle number was accompanied by striking changes in the appearance of the synaptic terminals and an increase in their mean circumference. Coated pits and membrane-bound cisternae were observed more frequently in synapses and varicosities of depolarized neurons than in terminals of resting neurons. These morphological changes were not seen when the neurons were depolarized in the presence of Co2+, consistent with the Ca2+-dependence of transmitter release from these neurons. In freeze-fracture replicas of depolarized neurons, numerous dimples were observed in the cytoplasmic leaflet of synapses and varicosities, adjacent to large 12-14 nm particles. After a period of recovery in 5 mM K+ medium, the number of synaptic vesicles and the shape of synaptic terminals returned to normal. When horseradish peroxidase (HRP) was included in the medium as an extracellular tracer during depolarization and recovery, a significant proportion of small, synaptic vesicles contained reaction product. Label was also present in coated vesicles and cisternae. Neurons which were depolarized in medium containing Co2+ or were exposed to HRP without depolarization contained few labelled synaptic vesicles. The proportion of labelled vesicles was not significantly different in synapses and varicosities, nor did it vary consistently with the transmitter identity of the neurons. These observations are consistent with the hypothesis that transmitter release occurs from varicosities as well as from synapses of postganglionic sympathetic neurons by exocytosis of the small synaptic vesicles, and that at least some new vesicles are formed from the nerve terminal membrane.

Animals↗

PubMed1996

Structures of the extracellular domain of the type I tumor necrosis factor receptor.

BACKGROUND: Tumor necrosis factor (TNF) is a powerful cytokine that is involved in immune and pro-inflammatory responses. Two TNF receptors that belong to the cysteine-rich low affinity nerve growth factor receptor family (TNF-R1 and TNF-R2) are the sole mediators of TNF signalling. Signalling is thought to occur when a trimer of TNF binds to the extracellular domains of two or three receptor molecules, which permits aggregation and activation of the cytoplasmic domains. The complex is then internalized within an endocytic vesicle, whereupon it dissociates at low pH. Structure of the soluble extracellular domain of the receptor (sTNF-R1) both in the unliganded and TNF-bound state have previously been determined. In both instances, the fourth subdomain of the receptor was found to be partly disordered. In the unliganded state at pH 7.5, the extracellular domain forms two distinct types of dimer, parallel and antiparallel; the antiparallel dimer occludes the TNF-binding. RESULTS: We have determined the structure of sTNF-R1 in two crystal forms in high salt at pH 3.7. The orthorhombic crystals diffract to 1.85 ånd the entire polypeptide is well ordered. In contrast, the C-terminal 32 residues are disordered in the hexagonal crystals. In the orthorhombic form, these residues exhibit a topology and disulphide connectivity that differs from the other three cysteine-rich domains in the molecule. In both forms, the interface is considerably more extensive than that used in complex formation with LTalpha. This 'low pH' dimer is different from both of the dimers observed in crystals grown at pH 7.5. CONCLUSIONS: The occurrence of the antiparallel dimers in both low pH crystal forms suggest that they are not an artefact of crystal packing. Such dimers may form in the low pH environment of the endosome. Because the dimer contact surface occludes the TNF-binding site, formation of this dimer would dissociate the TNF-receptor complex within the endosome. Three of the four cysteine-rich domains of TNF-R1 are constructed from two distinct structural modules, termed A1 and B2. The fourth subdomain comprises an A1 module followed by an unusual C2 module. Although the orientation of these modules with respect to each other is sensitive to crystal packing, ligand binding, pH and ionic strength, the modules are structurally well conserved between and within the known sTNF-R1 structures.

Antigens, CD↗

PubMed2001

Changes in phospholipid extractability and composition accompany mineralization of chicken growth plate cartilage matrix vesicles.

Matrix vesicles are lipid bilayer-enclosed structures that initiate extracellular mineral formation. Little attention has been given to how newly formed mineral interacts with the lipid constituents and then emerges from the lumen. To explore whether specific lipids bind to the incipient mineral and if breakdown of the membrane is involved, we analyzed changes in lipid composition and extractability during vesicle-induced calcification. Isolated matrix vesicles were incubated in synthetic cartilage lymph to induce mineral formation. At various times, samples of the lipids were taken for analysis, extracted both before and after demineralization to remove deposited mineral. Phosphatidylserine and phosphatidylinositol both rapidly disappeared from extracts made before decalcification, indicating rapid degradation. However, extracts made after demineralization revealed that phosphatidylserine had become complexed with newly forming mineral. Concomitantly, its levels actually increased, apparently by base-exchange with phosphatidylethanolamine. Though partially complexed with the mineral, phosphatidylinositol was nevertheless rapidly broken down. Sphingomyelin and phosphatidylethanolamine also underwent rapid breakdown, but phosphatidylcholine was degraded more slowly, all accompanied by a buildup of free fatty acids. The data indicate that phosphatidylserine forms complexes that accompany mineral formation, while degradation of other membrane phospholipids apparently enables egress of crystalline mineral from the vesicle lumen.

Animals↗

PubMed1986

Differential subcellular distribution of bioactive human chorionic gonadotropin in pseudopregnant rat ovary.

The subcellular metabolism of internalized hCG was examined by monitoring the distribution of bioactive and immunoreactive hCG in subcellular fractions of pseudopregnant rat ovaries. Homogenates of ovaries from rats injected with 1.0 microgram (12.8 IU; bioassay) hCG were fractionated on self-generating Percoll gradients into three hCG-containing compartments: soluble proteins (cytosolic fraction), a combined plasma membrane/prelysosomal vesicle fraction, and lysosomes. The hCG level in each fraction was measured by RIA and in vitro bioassay. When necessary, receptor-bound hCG was dissociated at low pH before assay. Levels of cytosolic hCG were highest 1-3 h after injection, attaining peak immunoreactive concentrations of 18 ng/ovarian pair. Cytosolic hormone was not primarily derived from nonspecific trapping of serum or interstitial fluid, because after 1.0 microgram [125I]iodobovine gamma-globulin was injected into rats, cytosolic globulin levels (nanograms per ovarian pair) were approximately 7-10 times lower than those of hCG. Cytosolic hCG retained significant bioactivity for at least 10 h after hCG stimulation. Peak immunoreactive hCG levels associated with the plasma membrane/prelysosomal fraction were 82 ng/ovarian pair between 3 and 6 h after hCG injection, and hormone associated with that fraction retained the highest bioactivity of the three fractions examined. Peak lysosomal hCG levels reached 55 ng/ovarian pair 10 and 14 h after hCG stimulation, but lysosomal hCG was not bioactive. These results suggest that the lysosomal compartment is a major pathway for hCG inactivation. A nonlysosomal pathway for hCG inactivation may exist, because the cytosolic compartment contained partially inactivated hormone that did not appear to be of lysosomal origin. Cytosolic hCG may reflect hormone delivered to the cell cytoplasm or to the extracellular fluid that is either modified within prelysosomal vesicles or is degraded subsequently by nonlysosomal proteases.

Animals↗